Diabetes is characterized by impaired glucose metabolism manifesting itself, among other things, by an elevated blood glucose level in the diabetic patient. Underlying defects lead to a classification of diabetes into two major groups: type 1 diabetes, or insulin dependent diabetes mellitus (IDDM), which arises when patients lack β-cells producing insulin in their pancreatic glands, and type 2 diabetes, or non-insulin dependent diabetes mellitus (NIDDM), which occurs in patients with an impaired β-cell function and alterations in insulin action.
Type 1 diabetic patients are currently treated with insulin, while the majority of type 2 diabetic patients are treated with agents that stimulate β-cell function or with agents that enhance the tissue sensitivity of the patients towards insulin. Over time almost one-half of type 2 diabetic subjects lose their response to these agents and then must be placed on insulin therapy. The drugs presently used to treat type 2 diabetes are described below.
Alpha-glucosidase inhibitors (e.g., Precose®, Voglibose™, and Miglitol®) reduce the excursion of postprandial glucose by delaying the absorption of glucose from the gut. These drugs are safe and provide treatment for mild to moderately affected diabetic subjects. However, gastrointestinal side effects have been reported in the literature.
Insulin sensitizers are drugs that enhance the body's response to insulin. Thiozolidinediones such as Avandia™ (rosiglitazone) and Actos™ activate the peroxisome proliferator-activated receptor (PPAR) gamma subtype and modulate the activity of a set of genes that have not been well described. Rezulin™ (troglitazone), the first drug in this class, was withdrawn because elevated liver enzyme levels and drug induced hepatotoxicity. These hepatic effects do not appear to be a significant problem in patients using Avandia™ and Actos™. Even so, liver enzyme testing is recommended every 2 months in the first year of therapy and periodically thereafter. Avandia™ and Actos™ seem to be associated with fluid retention and edema. Avandia™ is not indicated for use with insulin because of concern about congestive heart failure. Another potential side effect is weight gain.
Insulin secretagogues (e.g., sulfonylureas (SFUs) and other agents that act by the ATP-dependent K+ channel) are another drug type presently used to treat type 2 diabetes. SFUs are standard therapy for type 2 diabetics that have mild to moderate fasting glycemia. The SFUs have limitations that include a potential for inducing hypoglycemia, weight gain, and high primary and secondary failure rates. Ten to 20% of initially treated patients fail to show a significant treatment effect (primary failure). Secondary failure is demonstrated by an additional 20-30% loss of treatment effect after six months on an SFU. Insulin treatment is required in 50% of the SFU responders after 5-7 years of therapy (Scheen, et al., Diabetes Res. Clin. Pract. 6:533-543, 1989).
Glucophage™ (metformin HCl) is a biguanide that lowers blood glucose by decreasing hepatic glucose output and increasing peripheral glucose uptake and utilization. The drug is effective at lowering blood glucose in mildly and moderately affected subjects and does not have the side effects of weight gain or the potential to induce hypoglycemia. However, Glucophage™ has a number of side effects including gastrointestinal disturbances and lactic acidosis. Glucophage™ is contraindicated in diabetics over the age of 70 and in subjects with impairment in renal or liver function. Finally, Glucophage™ has the same primary and secondary failure rates as the SFUs.
Insulin treatment is instituted after diet, exercise, and oral medications have failed to adequately control blood glucose. This treatment has the drawbacks that it is an injectable, that it can produce hypoglycemia, and that it causes weight gain.
Because of the problems with current treatments, new therapies to treat type 2 diabetes are needed. In particular, new treatments to retain normal (glucose-dependent) insulin secretion are needed. Such new drugs should have the following characteristics: dependent on glucose for promoting insulin secretion (i.e., produce insulin secretion only in the presence of elevated blood glucose); low primary and secondary failure rates; and preserve islet cell function. The strategy to develop the new therapy disclosed herein is based on the cyclic adenosine monophosphate (cAMP) signaling mechanism and its effects on insulin secretion.
Cyclic AMP is a major regulator of the insulin secretion process. Elevation of this signaling molecule promotes the closure of the K+ channels following the activation of protein kinase A pathway. Closure of the K+ channels causes cell depolarization and subsequent opening of Ca++ channels, which in turn leads to exocytosis of insulin granules. Little if any effects on insulin secretion occurs in the absence of low glucose concentrations (Weinhaus, et al., Diabetes 47:1426-1435, 1998). Secretagogues like pituitary adenylate cyclase activating peptide (“PACAP”) and GLP-1 (glucagon-like peptide 1) use the cAMP system to regulate insulin secretion in a glucose-dependent fashion (Komatsu, et al., Diabetes 46:1928-1938, 1997; Filipsson, et al., Diabetes 50:1959-1969, 2001; Drucker, Endocrinology 142:521-527, 2001). Insulin secretagogues working through the elevation of cAMP such as GLP-1 and PACAP is also able to enhance insulin synthesis in addition to insulin release (Skoglund, et al., Diabetes 49:1156-1164, 2000; Borboni, et al., Endocrinology 140:5530-5537, 1999).
PACAP is a potent stimulator of glucose-dependent insulin secretion from pancreatic β-cells. Three different PACAP receptor types (PAC1, VPAC1, and VPAC2) have been described (Harmar, et al., Pharmacol. Reviews 50:265-270, 1998; Vaudry, et al., Pharmacol. Reviews 52:269-324, 2000). PACAP displays no receptor selectivities, having comparable activities and potencies at all three-receptors. PAC1 is located predominately in the CNS, whereas VPAC1 and VPAC2 are more widely distributed. VPAC1 is located in the CNS as well as in liver, lungs, and intestine. VPAC2 is located in the CNS, pancreas, skeletal muscle, heart, kidney, adipose tissue, testis, and stomach. Recent work argues that VPAC2 is responsible for the insulin secretion from β-cells (Inagaki, et al., Proc. Natl. Acad. Sci. USA 91:2679-2683, 1994; Tsutsumi, et al., Diabetes 51:1453-1460, 2002). This insulinotropic action of PACAP is mediated by the GTP binding protein Gs. Accumulation of intracellular cAMP in turn activates the nonselective cation channels in β-cells increasing [Ca++], and promotes exocytosis of insulin-containing secretory granules.
PACAP is the newest member of the superfamily of metabolic, neuroendocrine, and neurotransmitter peptide hormones that exert their action through the cAMP-mediated signal transduction pathway (Arimura, Regul. Peptides 37:287-303, 1992). The biologically active peptides are released from the biosynthetic precursor in two molecular forms, either as a 38-amino acid peptide (PACAP-38) and/or as a 27-amino acid peptide (PACAP-27) with an amidated carboxyl termini (Arimura, supra).
The highest concentrations of the two forms of the peptide are found in the brain and testis (Arimura, supra). The shorter form of the peptide, PACAP-27, shows 68% structural homology to vasoactive intestinal polypeptide (VIP). However, the distribution of PACAP and VIP in the central nervous system suggests that these structurally related peptides have distinct neurotransmitter functions (Koves, et al., Neuroendocrinology 54:159-169, 1991).
Recent studies have demonstrated diverse biological effects of PACAP-38, from a role in reproduction (McArdle, Endocrinology 135:815-817, 1994) to an ability to stimulate insulin secretion (Yada, et al., J. Biol. Chem. 269:1290-1293, 1994). In addition, PACAP appears to play a role in hormonal regulation of lipid and carbohydrate metabolism (Gray, et al., Mol. Endrocrinol. 15:173947, 2001); circadian function (Harmar, et al., Cell 109: 497-508, 2002); and the immune system, growth, energy homeostasis, and male reproductive function (Asnicar, et al., Endrocrinol. 143:3994-4006, 2002); regulation of appetite (Tachibana, et al., Neurosci. Lett. 339:203-206, 2003); as well as acute and chronic inflammatory diseases, septic shock, and autoimmune diseases (e.g., systemic lupus erythematosus) (Pozo, Trends Mol. Med. 9:211-217, 2003).
Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide that was first isolated from hog upper small intestine (Said and Mutt, Science 169:1217-1218, 1970; U.S. Pat. No. 3,879,371). This peptide belongs to a family of structurally-related, small polypeptides that includes helodermin, secretin, the somatostatins, and glucagon. The biological effects of VIP are mediated by the activation of membrane-bound receptor proteins that are coupled to the intracellular cAMP signaling system. These receptors were originally known as VIP-R1 and VIP-R2, however, they were later found to be the same receptors as VPAC1 and VPAC2. VIP displays comparable activities and potencies at VPAC1 and VPAC2.
To improve the stability of VIP in human lung fluid, Bolin, et al., (Biopolymers 37:57-66, 1995) made a series of VIP variants designed to enhance the helical propensity of this peptide and reduce proteolytic degradation. Substitutions were focused on positions 8, 12, 17, and 25-28, which were implicated to be unimportant for receptor binding. Moreover, the “GGT” sequence was tagged onto the C-terminus of VIP muteins with the hope of more effectively capping the helix. Finally, to further stabilize the helix, several cyclic variants were synthesized (U.S. Pat. No. 5,677,419). Although these efforts were not directed toward receptor selectivity, they yielded two analogs that have greater than 100-fold VPAC2 selectivity (Gourlet, et al., Peptides 18:403-408, 1997; Xia, et al., J. Pharmacol. Exp. Ther., 281:629-633, 1997).
GLP-1 is released from the intestinal L-cell after a meal and functions as an incretin hormone (i.e., it potentiates glucose-induced insulin release from the pancreatic β-cell). It is a 37-amino acid peptide that is differentially expressed by the glucagon gene, depending upon tissue type. The clinical data that support the beneficial effect of raising cAMP levels in β-cells have been collected with GLP-1. Infusions of GLP-1 in poorly controlled type 2 diabetics normalized their fasting blood glucose levels (Gutniak, et al., New Eng. J. Med. 326:1316-1322, 1992) and with longer infusions improved the β-cell function to those of normal subjects (Rachman, et al., Diabetes 45:1524-1530, 1996). A recent report has shown that GLP-1 improves the ability of β-cells to respond to glucose in subjects with impaired glucose tolerance (Byrne, et al., Diabetes 47:1259-1265, 1998). All of these effects, however, are short-lived because of the short half-life of the peptide.
Amylin Pharmaceuticals is conducting Phase III trials with Exendin 4™ (AC2993), a 39 amino acid peptide originally identified in Gila Monster. Amylin has reported that clinical studies demonstrated improved glycemic control in type 2 diabetic patients treated with Exendin 4™. However, the incidence of nausea and vomiting was significant.
Applicants disclosed novel polypeptides that function in vivo as agonists of the VPAC2 receptor in WO 01/23420, the specification of which is incorporated herein in its entirety, and in particular, Applicants disclosed a VPAC2 agonist identified as R3P66. The polypeptides described therein, including R3P66, however, are not suitable commercial candidates given stability issues associated with the polypeptides in formulation, as well as issues with the polypeptides' short half-life.
There exists a need for improved peptides that have the glucose-dependent insulin secretagogue activity of PACAP, GLP-1, or Exendin 4™, but with fewer side-effects, and preferably which are stable in formulation and have long plasma half-lives. Furthermore, tighter control of plasma glucose levels may prevent long-term diabetic complications. Thus, new diabetic drugs should provide an improved quality of life for patients.